Nearly all licensed vaccines protect through antibodies rather than cell-mediated immunity. A critical aspect of vaccine-induced serological protection is the duration of antibody titer post-boost. Our basic understanding of T-cell dependent antibody responses involves presentation of antigens by dendritic cells to specific CD4+ T cells, which then proliferate and differentiate into helper T cells (Tfh) that engage antigen-engaged B cells that have moved to the T-B border of secondary lymphoid tissues (spleen or lymph node). These interactions can generate a population of short-lived, high-rate antibody secreting cells (the extrafollicular antibody response), which contributes to acute host defense but not to long-lived protection. The latter involves migration of antigen-activated T cells and the associated antigen-specific B cells into the B cell follicle where they set up the germinal center (GC) reaction. Here continued T-B interaction leads to somatic hypermutation and isotype class switching, producing antibodies of higher affinity and different effector class, while generating both memory B cells and also plasmablasts that can become long-lived plasma cells (LLPC) if they reach the proper niche, which is mainly located in the bone marrow. While this general outline is well-established, the molecular signals that guide the engagement of T and B cells to generate a maximally productive response, the role of Tfh cells in determining the choice between memory B cells and LLPCs, and what determines how plasmablasts become LLPCs remain unclear. This project is a collaborative effort among laboratories with expertise in vaccine development, adjuvant function, and cellular immunology that aims to examine how variations in the quality and quantity of Tfh, stimuli for B cells, and GC output contribute to the magnitude and durability of antibody responses post-vaccination. In FY16, we made significant progress towards evaluating the contribution of carrier determinants and adjuvants to promoting high-titer, long-lived antibody responses against Pfs25, a leading TBV candidate for Plasmodium falciparum. We determined that both adjuvants and carrier proteins influence the magnitude and capacity of Pfs25-specific humoral responses to remain above a protective level. Additionally, we found that a liposomal adjuvant formulation with a TLR4 agonist and QS21, GLA-LSQ, profoundly impacted the magnitude of the Tfh and LLPC response against Pfs25, an effect that was further enhanced following conjugation to antigenic carrier proteins. Importantly, this adjuvant-dependent Tfh cell priming correlated with a large LLPC response and durable, functional antibody response. We have prepared a manuscript summarizing this work and obtained legal approval from the Infectious Disease Research Institute (IDRI), the company that supplies the GLA-LSQ adjuvant, to submit this work to Scientific Reports in August 2016. In addition to our collaboration with IDRI, we established a Research Collaboration Agreement with the Walter Reed Army Institute of Research (WRAIR) to obtain an unrestricted supply of potent adjuvants with clinical potential. We are using these Army Liposome Formulation (ALF)-based adjuvants, in combination with the carrier-specific tetramers we developed in FY15, to relate Tfh quality and quantity to vaccine outcome. Our preliminary data suggest that Tfh quality is a defining factor regulating the size of the GC response. With regards to Tfh quality, we are particularly interested in whether different adjuvants promote CD4+ T cells to adopt a suppressive T follicular regulatory (Tfr) phenotype and, if so, where and when do these differentiation events occur in lymphoid organs. To address these questions, we are employing multi-parameter flow cytometry and advanced imaging techniques to quantify the kinetics and location of Tfh and Tfr differentiation following vaccination. We found that the ALFQ adjuvant induced a higher Tfh:Tfr cell ratio as compared to two alum containing adjuvants, alhydrogel and ALFQA. These qualitative differences in T helper differentiation correlated with larger GC and plasmablast responses after secondary immunization. Interestingly, Tfr cells were nearly absent from active GC reactions and were, instead, found at the T-B border in all the immunization groups examined, raising several questions about how Tfr cells regulate Tfh and GC B cells. The data generated by these rodent experiments will be extended to NHP studies focused on generating durable antibody responses with transmission-blocking activity. A key aim of this project is the identification of vaccine formulations that will increase the duration of the antibody response against malaria vaccine candidates. Our prototype target malaria antigen is Pfs25, which is undergoing Phase 1 trials in humans as a Pichia-expressed recombinant protein conjugated to the carrier protein ExoProtein A (EPA) expressed in E. coli with a molar ratio of 3:1, and formulated with the commercially available adjuvant Alhydrogel: Pfs25-EPA/Alhydrogel. Pfs25-EPA/Alhydrogel is undergoing Phase 1 trials in malaria-nave volunteers in the US (dose-escalating trial) and malaria-experienced volunteers in Mali (dose-escalating; double-blinded; placebo-controlled trial). Sera collected from volunteers in the US and Mali are being assessed for seroreactivity by standardized ELISA, and transmission-blocking antibody activity is measured in membrane feeding assays. Serum antibody levels in either assay are being measured before and after each vaccine dose, and then periodically thereafter to assess the duration of the antibody response, including the functional antibody response. In Mali, mosquitoes are fed directly on vaccinees to determine their infectivity/malaria transmission potential, and this will be related to the antibody measurements. The Pfs25-EPA/Alhydrogel product will be a benchmark against which we will compare novel Pfs25 products and formulations in our animal studies. Our animal studies of adjuvants to date support our plan to test Pfs25-based conjugate vaccines using alternative adjuvants in humans, and our initial focus is on the commercial product AS01 from GSK and similar liposomal formulations from IDRI and WRAIR that have not been in the clinic.